Kleefstra syndrome (KLEFS) is a rare inherited neurodevelopmental disorder (NDD) characterized by neuropsychiatric anomalies, developmental delay (DD), mild-to-severe intellectual disability (ID), childhood hypotonia, speech delay, and craniofacial abnormalities (1, 2). Globally, the prevalence stands between 1:25,000 and 1:35,000 individuals. KLEFS is primarily caused by a heterozygous microdeletion encompassing the Euchromatin Histone Methyltransferase 1 (EHMT1) gene on chromosome 9q34.3 or a pathogenic variant in the EHMT1 gene (3, 4). Additionally, pathogenic variants in KMT2C have been found in patients with a clinical diagnosis of Kleefstra syndrome-2. KMT2C (KMT2C; MIM 606833, NM_170606) is located on chromosome 7q36 and consists of 59 exons, encoding lysine-N-methyltransferase 2C, which catalyzes histone 3 lysine 4 (H3K4) methylation and epigenetically regulates gene transcription through chromatin modifications (5). Dysregulated H3K4 methylation can cause ID, epilepsy, autism spectrum disorder (ASD), schizophrenia, and other neurodevelopmental and neuropsychiatric disorders (6, 7). Recently, novel or de novo pathogenic variants in the KMT2C gene have been reported to cause a variety of developmental abnormalities, including ID, ASD, schizophrenia, and non-syndromic primary tooth eruption failure (8–13). To date, only 13 cases of KLEFS2 have been reported in the literature (8, 14–18). Currently, there is no clear association between the clinical phenotype and variant sites. Thus, additional reports would help us better understand the phenotype spectrum of this disease and explore the relationship between genotypes and phenotypes.

In this study, we present five additional patients diagnosed with Kleefstra syndrome-2 by whole-exome sequencing with a highly heterogeneous phenotype, including mild-to-moderate ID, facial dysmorphism, language and motor delays, short stature, weight loss, microcephaly, and behavioral abnormalities. Molecular analyses identified five novel KMT2C variants. Additionally, we have reviewed the literature to summarize the genotypes, phenotypes, and clinical features of KLEFS2.

KMT2C, also known as MLL3, is a histone lysine methyltransferase enzyme that plays an important role in modifying histone proteins (6, 8, 20, 21). It is involved in H3K4me1 and H3K4me3 marks related to active enhancers or transcriptionally active regions, respectively (22, 23). KMT2C is part of the protein complex associated with Set1 (COMPASS complex), which plays a vital role in eukaryotic developmental signaling pathways (24–26). KMT2C knockout mice display ASD-like repetitive behaviors, social deficits, and ID (27). In human, variants in KMT2C have been reported to be associated with ASD-like behaviors and Kleefstra syndrome-2 (18, 21, 25, 28–32). Notably, only 13 patients have been reported with variants in KMT2C. Among these patients, only one long-term report showed a long-term persistent phenotype. Therefore, the phenotypes associated with KMT2C mutations remain incompletely characterized. In this study, we found five KMT2C variants related to neurodevelopmental abnormalities. The patients exhibited common disease phenotypes, including mild-to-moderate ID and DD, failure to thrive, facial dysmorphism, speech delay, hypotonia, feeding difficulties, short stature, weight loss, microcephaly, and behavioral abnormalities.

In the patients described in this study, five variants were identified in KMT2C, including one non-sense variant and four frameshift variants. In silico, these null variants may cause a premature termination codon or a translational frameshift, leading to significantly reduced truncated proteins and markedly reduced mRNA levels due to NMD degradation. These variants were assessed as likely pathogenic or pathogenic according to the ACMG/AMP guidelines (Table 2).

Patient 5 harbors a potentially pathogenic heterozygous variant in the KMT2C gene inherited from his mother. Inconsistent phenotypes were observed in patient 5 and his mother. Patient 5 manifested a more extensive and severe phenotype, including delayed motor development, severe language impairment, moderate intellectual disability, autistic behaviors, ADHD, and dysmorphic facial features, while his mother exhibited only a mild cognitive impairment. Consequently, these findings suggest a high degree of variability in the clinical features among individuals with heterozygous mutations in the KMT2C gene.

In the current study, we describe the genotype and clinical phenotype of five patients with variants in KMT2C, bringing the total number of reported individuals to 18. The list of reported variants of KMT2C and the clinical phenotype of our patients and the other reported patients are summarized in Table 3 and Supplementary Table S1 (8, 14–18). Except for one missense variant and one in-frame deletion, all reported variants were loss-of-function variants (LoF; including non-sense variants, splicing variants, frameshifts, in-frame, and microdeletions). Some common phenotypes were observed in patients with these variants. All 18 patients exhibited a range of intellectual disability from mild to severe; 88% of the patients (15/17) displayed diverse minor facial dysmorphisms, including midface hypoplasia, thick and everted lower lip, abnormal eyebrows (arched or prominent), nose abnormalities (saddle bridge, bulbous tip nose, and short nose), and eye abnormalities (deep set eyes, strabismus, ptosis, down-slanting palpebral fissures, and hypertelorism). Speech delay was observed in all patients (17/17), ranging from delayed speech to remaining non-verbal after the age of 15 years. Nearly all patients (14/15) presented with motor delay or dyskinesia. All patients learned to walk, but some did not learn to walk until after the age of 4 years. Behavioral abnormalities were also observed in most patients (13/16) and became more characteristic with age. These abnormalities included autism, ADHD, sleeping disorder, aggressiveness, hyperactivity, automutilation, and elective mutism. Hypotonia was common (11/16), with one patient exhibiting a broad gait and another showing developmental regression. Short stature (12/17) and weight loss (11/17) were the prevalent features, both falling 2–4 standard deviations below the mean for the general population. Aripiprazole medication effectively improved weight in patients with low body weight; two patients, including ours, showed significant weight gain after aripiprazole medication, particularly in abdominal fat. Relative microcephaly was observed in nine out of 18 patients, while macrocephaly was noted in two patients. Additionally, various other variable dysmorphic features, such as brain abnormalities, seizures, phenylketonuria, recurrent respiratory infections, cryptorchidism, bifid uvula, bilateral inguinal hernia, delayed puberty, hearing impairment, insensitivity to pain, constipation, slight telelia, ligamentous hyperlaxity, and eczema, were also observed.

Neurodevelopmental disorders caused by chromatin-regulated protein-encoding genes often exhibit overlapping clinical manifestations (33–35). Comparing the clinical manifestations of these different related disorders can enhance our understanding of the broad clinical spectrum of these diseases and the significance of WES in the diagnostic process (36, 37). Although some consistent clinical manifestations of KLEFS2 have been observed, we currently cannot establish a clear correlation between the genotype and the phenotype, which is attributed to the complexity and heterogeneity of the KLEFS2 phenotypes. The diverse phenotype observation indicates that variants occurring at different positions may have site-specific effects on the phenotypes. Moreover, in this cohort of 18 patients, the male-to-female ratio was 1:1. Some severe phenotypes are more likely to be observed in women, including severe ID (4/9), severe speech impairment (3/9), brain abnormalities (3/9), and macrocephaly (2/9), suggesting that sex may influence the outcome. The underrepresentation of patients in this cohort may reflect ascertainment bias, and further confirmation of the observations is expected as the number of patients increases.

The exact mechanisms underlying how these variants cause ID, facial dysmorphisms, language and motor delays, behavioral abnormalities, hypotonia, short stature and weight loss, and additional clinical symptoms remain unclear. The KMT2C gene is highly conserved from archaea to eukaryotes and is widely expressed in human tissues with high levels in the cerebellum of the developing and adult human brain (38, 39). This gene encodes an enzyme that monomethylates lysine 4 of histone H3 (H3K4me1) and epigenetically regulates many gene expressions in the brain through chromatin modification (40). In 2017, Koemans et al. found that KMT2C knockdown drosophila exhibited severe defects in memory formation (8). Moreover, they found that KMT2C binds to the promoters of many genes that play important roles in neuronal processes (6). In vitro experiments show that reduced KMT2C expression leads to a decrease in the overall level of histone tail modifications H3K4me1 and H3K4me3 (6, 41). Furthermore, rare KMT2C variants have been associated with Kleefstra syndrome, ID, ASD, schizophrenia, non-syndromic primary failure of tooth eruption, and a family with colorectal cancer and acute myeloid leukemia (8, 14–18, 42). These findings suggest that the KMT2C variant has pleiotropic effects. Functional studies of these variants are needed to fully understand KLEFS2 and its mechanisms of action.

In conclusion, we present detailed clinical and phenotypic information on five additional unrelated Chinese patients with five novel heterozygous KMT2C variants, expanding the mutational spectrum of KLEFS2. This is the first report of the c.9232_9247delCAGCGATCAGAACCGT (p.Gln3078fs^*13), c.5068dupA (p.Arg1690fs^*10), c.10815_10819delAAGAA (p.Lys3605fs^*7), and c.6911_6912insA (p.Met2304fs^*8) variants in the KMT2C gene. Our study also indicates that there is currently no significant correlation between the genotype and phenotype, providing a foundation for clinical diagnosis and genetic counseling for KLEFS2.

The datasets presented in this article are not readily available because of ethical and privacy restrictions. Requests to access the datasets should be directed to the corresponding author.

The studies involving humans were approved by the Institutional Review Board and Ethics Committee of Guangxi Maternal and Child Health Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants' legal guardians/next of kin. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

QY: Conceptualization, Data curation, Formal analysis, Funding acquisition, Software, Writing—original draft, Writing—review & editing. QZ: Data curation, Methodology, Validation, Writing—review & editing. SheY: Writing—review & editing. SZ: Writing—review & editing. ShaY: Writing—review & editing. XZ: Writing—review & editing. ZQ: Writing—review & editing. BC: Writing—review & editing. JL: Writing—review & editing.